key: cord-0802199-qsdc267u
authors: Ruan, Zijing; Liu, Chao; Guo, Yuting; He, Zhenqing; Huang, Xinhe; Jia, Xu; Yang, Tai
title: SARS‐CoV‐2 and SARS‐CoV: Virtual screening of potential inhibitors targeting RNA‐dependent RNA polymerase activity (NSP12)
date: 2020-07-09
journal: J Med Virol
DOI: 10.1002/jmv.26222
sha: fdd38eb9a5d39bad8a7457c61aced8b7b190821a
doc_id: 802199
cord_uid: qsdc267u

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the harm caused by coronaviruses to the world cannot be underestimated. Recently, a novel coronavirus (severe acute respiratory syndrome coronavirus‐2 [SARS‐CoV‐2]) initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has infected more than six million people worldwide by 21 June 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 200 countries around the world. However, the effective drug has not yet been officially licensed or approved to treat SARS‐Cov‐2 and SARS‐Cov infection. NSP12‐NSP7‐NSP8 complex of SARS‐CoV‐2 or SARS‐CoV, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12‐NSP7‐NSP8 complex (PDB ID: 7BW4) structure of SARS‐CoV‐2 and the NSP12‐NSP7‐NSP8 complex (PDB ID: 6NUR) structure of SARS‐CoV, NSP12‐NSP7 interface model, and NSP12‐NSP8 interface model were established for virtual screening in the present study. Eight compounds (Nilotinib, Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All eight compounds can combine well with NSP12‐NSP7‐NSP8 in the crystal structure, providing drug candidates for the treatment and prevention of coronavirus disease 2019 and SARS.

implemented strict epidemic prevention and control measures across the country to avoid a larger-scale epidemic. 7 However, the mortality rate of coronavirus disease 2019 caused by SARS-CoV-2 is much higher than that of SARS at present. 4 The virus now presents in more than 200 countries. Even now though the SARS-CoV no longer appears on a large scale, the infectious power and harmful effects of SARS-CoV-2 and SARS-CoV should not be underestimated. To date, there are no officially licensed or approved drugs against this novel coronavirus and SARS-CoV. There is an urgent need to find new targets for the development of anti-SARS-CoV-2 and anti-SARS-CoV agents.

ORF1a and ORF1b at the 5′-terminus of the coronavirus (CoV) genomes encode polyprotein 1a and polyprotein 1b, the two proteins could be cleaved into 16 nonstructural proteins (NSPs), which are essential for viral replication and transcription, thus being regarded as a potential virulence factor and a target for CoV. 8, 9 Among these NSPs, the NSP12 subunit is the essential RdRp (RNA-dependent RNA polymerase) of the coronavirus replicative machinery, which was even able to extend a homopolymeric primer-template substrate by a few dozen nucleotides in vitro. 10, 11 The 3.1 Å cryo-EM structure of the SARS-CoV RNA polymerase NSP12 shows that it can bind with its essential cofactors NSP7 and NSP8. 12 The replication of the SARScoronavirus genome involves two RNA-dependent RdRps. The first is primer-dependent and associated with the NSP12, whereas the second is catalyzed by NSP8. NSP8 is capable of de novo initiating the replication process and has been proposed to operate as a primase. 13 In addition, NSP7, a component of the CoV replicase polyprotein, also participates in viral replication processed by binding to NSP12 as another primase. 13 The NSP12 needs to associate with NSP7 and NSP8 to activate its capability to replicate long RNA. 10 This elicits us to identify the particularly interesting compound disrupt the binding of NSP7 or NSP8 to NSP12, thus which could be used to inhibit the RdRp activity of NSP12, acting as novel antiviral agents and therapies of SARS-CoV-2 and SARS-CoV.

The amino acid sequence alignment revealed that the NSP12 (PDB ID: 7BW4) of SARS-CoV-2 shared 96.35% similarity with the NSP12 (PDB ID: 6NUR) of SARS ( Figure 1 ). In addition, comparative analyses of their deduced amino acid sequences revealed that NSP7 and NSP8 of SARS-CoV-2 shared 98.8% and 97.5% similarity with that of SARS-CoV respectively. The similarity of the target structure may also lead to similar antiviral drugs. Therefore, the NSP12 crystal structures of SARS-CoV-2 and SARS-CoV were used as the target proteins. The NSP7 and NSP8 binding pocket of NSP12 were designated as active sites for screening compounds. Computer virtual screening in known drug databases helps to quickly identify potential drug candidates for COVID-19 prevention and treatment. What's more, previous literature has shown that a number of virtuallyscreened compounds, such as ribavirin, lopinavir, and ritonavir, have proven to be effective in treating COVID-19. [14] [15] [16] [17] Here, through high-throughput screening methods using a pool of 30 000 small molecules, several potential drug candidates were identified for preventing the binding NSP7 or NSP8 to NSP12, suggesting further assessment of the anti-SARS-CoV-2 activity and anti-SARS-CoV activity of these compounds respectively in cell culture. 

The crystal structure of SARS-CoV-2 NSP12 (PDB ID: 7BW4) was used as the target protein of SARS-CoV-2. And the crystal structure of SARS NSP12 (PDB ID: 6NUR) was used as the target protein of SARS-CoV.

Vina1.1.2 was used to perform molecular docking. The docking boxes were set at the NSP12-NSP7 interface and NSP12-NSP8 interface ( Figure 1 ), respectively. The search exhaustiveness was set as 32, and the number of binding modes was set as 9. Other parameters were set as default. During docking, NSP7 (or NSP8) was removed from the complex and only NSP12 was left as a receptor.

F I G U R E 1 The docking boxes of the NSP12-NSP7 interface and NSP12-NSP8 interface. The yellow part is nsp12. The green part is nsp7. The cyan part is nsp8. The docking box on the left is NSP12-NSP7 interface. The docking box on the right is NSP12-NSP8 interface 2.4 | Binding free energy calculation

Each simulation system was immersed in a cubic box of TIP3P water with 10 Å distance from the solute. The Na + or Cl − was applied to neutralize the system. General Amber force field 15 and Amber ff14SB force field were used to parameterizing the ligand and protein respectively. A total of 10 000 steps of minimization with constraints (10 kcal/mol/Å2) on heavy atoms of complex, including 5000 steps of steepest descent minimization and 5000 steps of conjugate gradient minimization, were used to optimize each system. Then each system was heated to 300 K within 0. 

Nilotinib, a second-generation small-molecule tyrosine kinase inhibitor, is widely used in the treatment of chronic myeloid leukemia. 18 Studies have shown that Nilotinib has a potential antiviral effect. 19 Our docking results showed that Nilotinib was mainly combined with the interface between NSP12 and NSP7 of SARS-CoV-2 through van der Waals potential energy and hydrogen bonds, involving LYS-411 ( Figure 2A ). Nilotinib could bind to the interface active pockets of the SARS-CoV-2 NSP12 and NSP7 ( Figure 2B ).

Therefore, Nilotinib can be considered as a candidate drug for treating SARS-CoV-2 infection.

Saquinavir, the first HIV protease inhibitor was introduced into the 

Lonafarnib as a nonpeptidomimetic inhibitor of farnesyltransferase has been used for progeria. 23 

Olysio, HCV NS3/4A protease inhibitor approved for the treatment of genotype 1 chronic hepatitis C in combination with pegylated interferon and ribavirin. 28 Our docking results showed that the hydrogen bonds and van der Waals forces maintained upon the binding of Olysio and SARS-CoV-2 NSP12-NSP8 interface, involving VAL-330

and TYR-273 ( Figure 6A ). What is more, the hydrogen bonds involving VAL-214 and van der Waals forces maintained upon the binding of the Olysio and SARS-CoV NSP12-NSP8 interface ( Figure 6C ).

Olysio could also bind to the interface active pockets of the NSP12-NSP8 of SARS-CoV-2 and SARS-CoV ( Figures 6B and 6D) .

The binding model of Lonafarnib against SARS-CoV-2 and SARS-CoV NSP12-NSP7. A, Interactions between Lonafarnib (cyan) and associated residues (off-white) in the interface of the crystal structure for SARS-CoV-2. B, Binding models of Lonafarnib (cyan) in the SARS-CoV-2 NSP12-NSP7 protein interface pocket (white surface). C, Interactions between Lonafarnib (cyan) and associated residues (offwhite) in the interface of the crystal structure for SARS-CoV. D, Binding models of Lonafarnib (cyan) in the SARS-CoV NSP12-NSP7 protein interface pocket (white surface). SARS-CoV-2, severe acute respiratory syndrome coronavirus-2 F I G U R E 5 The binding model of Tegobuvir against SARS-CoV-2 and SARS-CoV. A, Interactions between Tegobuvir (cyan) and associated residues (off-white) in the interface of the crystal structure for SARS-CoV-2. B, Binding models of Tegobuvir (cyan) in the SARS-CoV-2 NSP12-NSP7 protein interface pocket (white surface). C, Interactions between Tegobuvir (cyan) and associated residues (off-white) in the interface of the crystal structure for SARS-CoV-2. D, Binding models of Tegobuvir (cyan) in the SARS-CoV-2 NSP12-NSP8 protein interface pocket (white surface). E, Interactions between Tegobuvir (cyan) and associated residues (offwhite) in the interface of the crystal structure for SARS-CoV. 

Cepharanthine, an alkaloid tetrandrine isolated from Stephania tetrandra was found to exert strong anticancer, anti-inflammatory, and antioxidant activities. 29 In addition, it shows in vitro inhibitory effect on Herpes simplex virus type 1 (HSV-1) infected cells. 30 

Filibuvir is an effective oral non-nucleoside HCV NS5B RdRp inhibitor which exhibits potent antiviral activity against subgenomic HCV replicons in cell culture assays and is a potential treatment of chronic HCV infection. 31, 32 Studies have shown that Filibuvir was well-tolerated and could be considered in combination with other antiviral drugs to achieve better safety and efficacy for chronic HCV. 31 Our docking results showed that the hydrogen bonds involving VAL-330 maintained upon the binding of Cepharanthine and SARS-CoV-2 NSP12-NSP8 interface, with additionally van der Waals forces ( Figure 8A ). Filibuvir was mainly combined with the SARS-CoV NSP12-NSP8 interface through van der Waals potential energy ( Figure 8C ). Filibuvir could bind to the interface active pockets of the SARS-CoV-2 and SARS-CoV NSP12-NSP8 (Figures 8B and 8D) . Thus, Filibuvir can be considered as a candidate drug for treating SARS-CoV-2 and SARS-CoV infection, providing evidence for further research.

F I G U R E 6 The binding model of Olysio against SARS-CoV-2 and SARS-CoV NSP12-NSP8. A, Interactions between Olysio (cyan) and associated residues (off-white) in the interface of the crystal structure for SARS-CoV-2. B, Binding models of Olysio (cyan) in the SARS-CoV-2 NSP12-NSP8 protein interface pocket (white surface). C, Interactions between Olysio (cyan) and associated residues (off-white) in the interface of the crystal structure for SARS-CoV-2. D, Binding models of Olysio (cyan) in the SARS-CoV-2 NSP12-NSP8 protein interface pocket (white surface). Numbers accompanying dashed yellow lines represent the interaction distance (Å, C). SARS-CoV-2, severe acute respiratory syndrome coronavirus-2

3.9 | Binding free energy calculated by MM/GBSA 

The authors declare that there are no conflict of interests.

RZ, YT, JX, and HX designed the study. RZ and LC performed most studies, but a few were carried out by GY and HZ. All authors contributed thoughts and advice. YT, JX, and HX did a literature search.

YT and JX collected the data. RZ. and LC analyzed and interpreted the data. RZ wrote the text, and the other authors contributed to the final text presentation. All authors have proved the submission. Abbreviation: SARS-CoV-2, severe acute respiratory syndrome coronavirus-2. a ΔE vdw = van der Waals energy terms; ΔE ele = electrostatic energy; ΔG gb = polar solvation free energy; ΔG np = nonpolar solvation free energy; ΔG cal = final estimated binding free energy calculated from the above terms (kcal/mol).

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How to cite this article

Virtual screening of potential inhibitors targeting RNA-dependent RNA polymerase activity (NSP12)